Multivalent MHC complex peptide fusion protein complex for stimulating specific T cell function

ABSTRACT

The present invention describes a soluble fusion protein composed of a plurality of major histocompatibility complex (MHC) molecules linked together by a stabilizing structure herein referred to as the “linker,” the MHC molecules being loaded with a specific peptide or peptides. Such fusion proteins can be used as a method for stimulating or inhibiting specific T cell clones expressing T cell receptors (TCR) restricted to the specific MHC-peptide combination. Such fusion proteins can thus be used as delivery systems to stimulate T cell immunity and as a treatment for diseases such as transplant rejection or autoimmunity.

BACKGROUND OF THE INVENTION

T cells mediate many immune responses, including transplant rejection,autoimmunity, viral infections, and tumor surveillance. T cellrecognition of peptide antigens occurs via the T cell receptor (TCR) andrequires that such antigen be presented to the TCR by a majorhistocompatibility complex (MHC) molecule, generally situated on thesurface of an antigen presenting cell. The peptide antigen is held bythe MHC molecule such that the T cell receptor recognizes the uniquestructure formed by the combination of the MHC molecule and the specificpeptide. Thus, only a small percentage of T cell clones react to a givenpeptide.

There are two major known types of MHC molecules: class I and class II.MHC class I molecules are composed of an alpha chain with 3 domains (α1,α2, and α3), as well as transmembrane and cytoplasmic domains. The α1and α2 domains are polymorphic. A non-polymorphic protein,β2-microglobulin, self associates with the alpha chain and is necessaryfor stable conformation. MHC class I molecules are widely distributedand are present on most nucleated cells.

MHC class II molecules are composed of an alpha chain and a beta chainthat self associate to form a heterodimer. Each chain has twoextracellular domains (α1, α2 and β1, β2), as well as transmembrane andintracellular domains. The α1 and β1 domains are polymorphic. MHC classII molecules are more restricted in distribution than are class Imolecules.

Polymorphisms in the MHC molecules, as well as the wide spectrum ofunique peptides that can associate with the MHC, result in an extremelydiverse recognition pattern such that a given MHC-peptide combination isonly recognized by a small percentage of T cell clones.

Present methods for modulating T cell function suffer from a number oflimitations including lack of specificity. For example, therapies forsuppressing T cell function (such as in autoimmunity or transplantrejection) cause generalized immunosuppression and may leave patients atrisk for developing life-threatening infections. The ultimate goal ofanti-T cell immunosuppressive therapy is to inhibit specific T cellalloreactive or autoreactive clones while leaving the majority of Tcells fully functional. Specific immunosuppressive therapy requirestargeting T cell clones recognizing specific MHC/peptide combinations.Several researchers have attempted to use soluble class I MHC moleculesto inhibit allogenic T cell responses in vitro or in vivo. In general,soluble class I molecules have not effectively inhibited alloreactive Tcell responses. Failure to observe inhibition of T cell function withsoluble MHC may relate to the requirement for divalency to induce T cellanergy.

Present therapies for enhancing T cell function (such as in certaininfections and malignancies) are often insufficient to induce anadequate immune response. Immunization with peptides alone has often notbeen successful at inducing a sufficient T cell response, since thepeptide is quickly degraded by peptidases.

Several reports indicate that divalency of the MHC molecules is criticalfor signal delivery to the T cell, including both activating andinhibitory signals. Further, T cell priming requires stimulation via theTCR and an additional second signal generally delivered by an antigenpresenting cell. In the absence of a second signal, T cellhyporesponsiveness results.

SUMMARY OF THE INVENTION

The present invention includes the process of creating a fusion proteinthat modulates T cell function in a peptide-specific manner, and thevarious methods by which the fusion protein modulates such function. Thepresent invention is premised on the realization that a fusion proteinwhich modulates specific T cell activity consists of three parts: (1) aplurality of MHC molecules; (2) a linker connecting the MHC molecules;and (3) a specific peptide or peptides loaded into the MHC molecules. Inparticular, the invention is directed to a fusion protein comprising aplurality of MHC molecules complexed to both a linker and to a selectedpeptide. The fusion protein targets the T cell receptor and modulates Tcell function. Methods of stimulating, inhibiting or destroying T cellsare provided by the fusion proteins. By constructing a fusion protein inwhich the linker allows delivery of a second signal, T cell stimulationresults in enhanced T cell immunity. By constructing a fusion protein inwhich the linker does not provide for delivery of a second signal, Tcell suppression results in immunosuppression. The fusion proteins canbe delivered in vivo as superior therapeutic agents for T cell-mediatedprocesses such as autoimmunity, infections, malignancies, andtransplantation rejection.

The objects and advantages of the present invention will be furtherappreciated in light of the following detailed description and drawingsin which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the PCR reactions used to forman MHC I IgG fusion protein.

FIG. 2 is a schematic representation of the PCR reactions used to forman MHC II IgG fusion protein.

DETAILED DESCRIPTION

The present invention includes the process of creating a fusion proteinthat modulates T cell function in a peptide-specific manner, and thevarious methods by which the fusion protein modulates such function. Thepresent invention is premised on the realization that a fusion proteinwhich modulates specific T cell activity consists of three parts: (1) aplurality of MHC molecules; (2) a linker connecting the MHC molecules;and (3) a specific peptide or peptides loaded into the MHC molecules. Inparticular, the invention is directed to a fusion protein comprising aplurality of MHC molecules complexed to both a linker and to a selectedpeptide. The fusion protein targets the T cell receptor and modulates Tcell function. Methods of stimulating inhibiting or destroying T cellsare provided by the fusion proteins.

The MHC molecules of the fusion protein can be either MHC class I or MHCclass II and can consist of the entire MHC chains, the extracellularportions of the chains, the peptide binding portion of the chains, orany other suitable fragment of MHC. Exemplary human MHC moleculesinclude HLA-A, HLA-B, HLA-C, DP, DQ and DR. Bivalency or multivalency ofthe MHC molecules is critical for signal delivery (either activation orinhibition signals) to the T cell. Therefore, the fusion protein of thepresent invention includes at least two identical MHC molecules attachedto a linker.

The linker of the fusion protein serves three functions. First, thelinker contributes the required bivalency or multivalency. Second, thelinker increases the half-life of the entire fusion protein in vivo.Third, the linker determines whether the fusion protein will activate orsuppress T cells. T cell priming requires stimulation via the TCR and anadditional second signal generally delivered by the antigen presentingcell. In the absence of a second signal, T cell hyporesponsivenessresults. By constructing a fusion protein in which the linker allowsdelivery of a second signal, T cell stimulation results in enhanced Tcell immunity. By constructing a fusion protein in which the linker doesnot provide for delivery of a second signal, T cell suppression resultsin immunosuppression.

A fusion protein with T cell stimulatory properties can be constructedby using a linker which allows for delivery of a second signal to the Tcell in addition to the signal delivered via the TCR. This can beaccomplished by using a linker that has binding affinity for a cellsurface structure on another cell, that cell being capable of deliveringa second signal to the T cell. Thus, the linker serves to bridge the Tcell and the other cell. By bringing the other cell into close proximityto the T cell, the other cell can deliver a second signal to the T cell.Examples include linkers that can bind to Fc receptors on other cellssuch as certain immunoglobulin chains or portions of immunoglobulinchains. Specific examples include IgG, IgA, IgD, IgE, and IgM. When animmunoglobulin is used, the entire protein is not required. For example,the immunoglobulin gene can be cleaved at the hinge region and only thegene encoding the hinge, CH2, and CH3 domains of the heavy chain is usedto form the fusion protein. The linker may bind other cell surfacestructures. For example, the linker can include a cognate moiety formany cell surface antigens which can serve as a bridge to bring thesecond cell into close proximity with the T cell. The linker might alsodeliver a second signal independently. For example, a linker withbinding affinity for the T cell antigen CD28 can deliver a secondsignal. Thus the linker contributes the required bivalency ormultivalency and determines whether the fusion protein will serve toenhance or suppress T cell function. In addition, the linker canincrease the half-life of the entire fusion protein in vivo.

A fusion protein with T cell inhibitory properties can be constructed byusing a linker that does not result in delivery of a second signal.Examples include Ig chains that do not bind Fc receptor, Ig F(ab′)₂fragments, a zinc finger motif, a leucine zipper, and non-biologicalmaterials. Examples of non-biological materials include plasticmicrobeads, or even a larger plastic member such as a plastic rod ortube, as well as other physiologically acceptable carriers which areimplantable in vivo.

The specific peptide of the fusion protein can be loaded into the MHCmolecules after the fusion protein has been made. The peptide may alsobe subsequently covalently attached to the MHC, for example by UVcross-linking. Alternatively, a peptide sequence can be incorporatedinto the DNA sequence encoding the fusion protein such that the peptideis loaded into the MHC molecules during generation of the fusionprotein. In the latter example, the peptide can be attached with atether, such as polylysine, which allows it to complex with the MHCportion of the fusion protein.

The specific peptides to be loaded into the MHC molecules are virtuallylimitless and are determined based on the desired application. Forexample, to enhance T cell immunity to viral, fungal and bacterialinfections, or to tumors, peptides from these sources can be used. Tosuppress T cell immunity in autoimmunity, autoreactive peptides can beused. To suppress T cell immunity to transplanted tissues, self peptideswhich are presented by alloantigen can be used.

Toxins, such as ricin and diphtheria toxin, and radioisotopes, may becomplexed to the fusion protein (for example, using5-methyl-2-iminothiolane) to kill the specific T cell clones. Thesetoxins can be chemically coupled to the linker or to the MHC portion ofthe fusion protein, or they can be incorporated into the DNA sequenceencoding the fusion protein such that the toxin is complexed to thefusion protein during generation of the fusion protein.

The fusion protein can be prepared by constructing a gene which encodesfor the production of the fusion protein. Alternatively, the componentsof the fusion protein can be assembled using chemical methods ofconjugation. Sources of the genes encoding the MHC molecules and thelinkers can be obtained from DNA databases such as GenBank, as well asfrom published scientific literature in the public domain. In the caseof MHC class I fusion proteins, the MHC fragment can be attached to thelinker and β2 microglobulin can be allowed to self-associate.Alternatively, the fusion protein gene can be constructed such that β2microglobulin is attached to the MHC fragment by a ether. In the case ofMHC class II fusion protein, either the alpha or the beta chain can beattached to the linker and the other chain can be allowed toself-associate. Alternatively, the fusion protein gene can beconstructed such that the alpha and beta chains are connected by atether. Peptides can be prepared by encoding them into the fusionprotein gene construct or, alternatively, with peptide synthesizersusing standard methodologies available to one of ordinary skill in theart. The resultant complete fusion proteins can be administered byinjection into the patient and can be repeated if necessary to provide aboosting reaction. Generally, the amount of fusion protein administeredby injection for therapeutic purposes would range from about 1 μg toabout 100 mg per kilogram body weight. With a solid linker, the fusionprotein could be injected if microparticles are used, or physicallyimplanted if a larger linker is used.

The construction and use of the fusion proteins of the present inventionis further explained and demonstrated by the following detailedexamples:

EXAMPLE 1 Design of a Class I Fusion Protein Design of a DivalentK^(b)/IgG₁

As shown in FIG. 1, constructs encoding the hinge, CH2 and CH3 regionsof mouse IgG, heavy chain and the extracellular domains (α1, α2, and α3)of the MHC class I (H-2K^(b)) molecule were individually amplified usingthe polymerase chain reaction (PCR). Primers 1 and 2 were used toamplify the MHC class I fragment. Primers 3 and 4 were used to amplifythe IgG₁ fragment. The 5′ end of primer 2 has sequence homology to the5′ end of the hinge region of IgG₁. The 5′ end of primer 3 has sequencehomology to the 3′ end of the α3 domain of K^(b). The products wereannealed in a subsequent PCR reaction using primers 1 and 4 to generatethe fusion gene and amplified by another round of PCR. The sequences ofthe PCR primers were:

Primer 1: G C G C A T C G A T A T G G T A C C G T G C A C G C T G C T(SEQ ID NO: 1);

Primer 2: C C C T G G G C A C C C A T C T C A G G G T G A G G G G C (SEQID NO: 2);

Primer 3: C C T G A G A T G G G T G C C C A G G G A T T G T G G T (SEQID NO: 3);

Primer 4: A A G C A T T C T A G A T C A T T T A C C A G G A G A G T G(SEQ ID NO: 4).

The final product was digested with restriction enzymes and ligated intothe expression vector pRcCMV, encoding the neomycin resistance gene.Escherichia coli strain DH5α was transformed and ampicillin resistantcolonies were selected. DNA from transformed colonies was extracted andthe entire fusion gene was sequenced. The fusion construct wastransiently transfected into COS-7 cells using calcium phosphateprecipitation. The plasmid pHuActβ2, encoding murine β2 microglobulinunder the control of the human β actin promoter, was cotransfected. Theresulting fusion protein was a soluble homodimer of 120 kd.

Stable transfectants were generated by electroporating (960 μF, 260 V, ∝resistance) J558L cells (ATCC) with 10 μg of the fusion protein plasmidand 10 μg of pHuActβ2. For a negative control, cells were transfectedwith 10 μg of pRcCMV without insert. Cells were grown for 24 hours andneomycin resistant cultures were selected by growing the cells in 900μg/ml G418.

Immunoprecipitation with Y3-sepharose. The monoclonal antibody Y3, whichrecognizes a conformational epitope of H-2K^(b), was conjugated tosepharose and used to immunoprecipitate ³⁵S-labeled supernatants fromthe stable transfectants. Immunoprecipitation with this monoclonalantibody yielded a 120 kDa homodimer, whereas negative control celllines had no protein precipitated by this monoclonal antibody. Thisresult indicated that the epitope recognized by Y3 is preserved in thefusion protein.

ELISAs. A Y3-based ELISA and an ELISA using a commercially availableanti-H-2K^(b) monoclonal antibody (recognizing an epitope distinct fromY3) was used to measure the presence of the fusion protein insupernatants derived from the stable transfectants. Supernatants fromcells expressing the fusion protein construct were reactive with both ofthe H-2K^(b)-specific monoclonal antibodies whereas control supernatantsshowed no reactivity with these antibodies. The binding of Y3 to thefusion protein was increased by loading the fusion protein with apeptide known to bind efficiently to H-2K^(b) (ovalbumin 257-264; ova).This result indicates that the fusion protein can be loaded with peptidewhich binds to H-2K^(b) efficiently.

Activation of a T-hybridoma using immobilized fusion protein. TheH-2K^(b)-restricted, ova-specific T-hybridoma B3.645, was cultured withfusion protein which was immobilized to polystyrene using an anti-IgG1antibody. The fusion protein was loaded with either ova 257-264 or acontrol peptide (vesicular stomatitis virus nuclear protein 52-59; vsv),which is known to bind to H-2K^(b). The T-hybridoma secreted interleukin2 (IL-2) only in response to the fusion protein which was loaded withova, but not the control vsv peptide. Control supernatant, containingova without fusion protein, also did not induce IL-2 secretion. Further,the fusion protein loaded with ova was not able to induce IL-2 secretionfrom a T-hybridoma (2B4) restricted to another MHC molecule. Theseresults indicate that the fusion protein was able to activate B3.645through the TCR, and that activation was peptide specific and MHCrestricted.

Activation of an H-2K^(b) restricted, ova-specific cytotoxic T cell(CTL) using immobilized fusion protein. Immobilized, ova-loaded fusionprotein was able to induce ova-specific H-2K^(b) restricted CTL tosecrete IL-3. In contrast, fusion protein loaded with vsv, or controlsupernatant containing ova alone, were not able to induce IL-3secretion. This result shows that the fusion protein activates a T cellline, in addition to a T-hybridoma. Additionally, it shows that thefusion protein has biological effects on CTL.

T cell anergy induced by immobilized fusion protein. B3.645 cells werecultured with ova-loaded immobilized fusion protein for twenty-fourhours. The cells were collected and rested for 3 days, at which timethey were re-exposed to ova-loaded immobilized fusion protein.Measurements of IL-2 indicated that B3.645 cells which had received aprimary stimulus of ova-loaded fusion protein were not able to respondto a subsequent stimulation with the ova-loaded fusion protein. Incontrast, if the primary stimulus was vsv-loaded fusion protein, theB3.645 cells were able to respond to a secondary stimulation ofova-loaded fusion protein. The anergy induced in this cell line was notdue to down-modulation of the TCR, as demonstrated by flow cytometryanalysis. These results show that the fusion protein is able to induceanergy in a T-hybridoma in a peptide specific manner.

Soluble fusion protein inhibits secretion of IL-2 from B3.645 inresponse to ova-loaded antigen presenting cells: B3.645 cells incubatedwith antigen presenting cells (EL4) pulsed with the ova peptide produceIL-2. Ova-loaded fusion protein was able to inhibit the secretion ofIL-2 from such ova-pulsed B3.645 T-hybridoma cells. This demonstratesthat soluble fusion protein loaded with the appropriate peptide preventsthe activation of the T-hybridoma.

Serum half life. One ml of ammonium sulfate concentrated culturesupernatant-containing fusion protein was injected intraperitoneallyinto C57BL/10 mice. Mice were bled at various intervals and sera weretested by ELISA. No drop in titers was noted over a 2-week observationperiod, indicating that the fusion protein was stable in vivo.

Suppression of skin allograft rejection. C57BL/10 (H-2k^(b)) mice weretreated with 1 ml of ammonium sulfate concentrated culturesupernatant-containing fusion protein. A skin graft from a B6.C-H^(bm1)donor (a congenic strain differing at the K locus) was then grafted.Skin graft rejection was delayed in mice treated with fusion protein,but not in controls.

EXAMPLE 2 Design of a Class II Fusion Protein Design of DivalentIA^(q)/IgG₃

Part I: Generation of soluble α-chain of IA^(q)

PCR was used to amplify the extracellular portion of the α-chain from acDNA clone. The 5′ primers were designed to incorporate either a Bgl IIor a Pst I restriction site for subconing into one of the pCMVexpression vectors. The 3′ primer was designed to incorporate a Sma Irestriction site. Sequences of the primers were:

1. alpha 5′ Bgl: 5′ AAAGATCTAGGATGCCGCGCAGCAGA 3′ (SEQ ID NO. 5)

2. alpha 5′ Pst: 5′ AACTGCAGAGGATGCCGCGCAGCAGA 3′ (SEQ ID NO. 6)

3. alpha 3′ Sma: 5′ AACCCGGGTTAAGTCTCTGTCAGCTC 3′ (SEQ ID NO. 7)

The cDNA was amplified with the primer sets: alpha 5′ Bgl and alpha 3′Sma or alpha 5′ Pst and alpha 3′ Sma. The final PCR products wereelectrophoresed through 1% agarose gels, stained with ethidium bromideand the appropriate size bands (650 bp) were excised. The DNA waspurified using the Gene Clean II Kit (BIO 101, Inc., Vista, Calif.)according to the manufacturer's instructions. Purified DNA was digestedwith the appropriate restriction enzymes (either Bgl II and Sma I or PstI and Sma I) and then ligated into the expression vector pCMV4 which hadbeen digested with Bgl II and Sma I or into pCMV8 which had beendigested with Pst I and Sma I. The ligations were transformed intocompetent Escherichia coli strain JM109 and ampicillin resistantcolonies were selected, DNA was prepared, and the entire gene wassequenced to ensure no spurious mutations were introduced during thePCR.

Part II: Generation of the IgG₃/β-chain Fusion Gene

As shown in FIG. 2, the fusion gene was generated through a series ofnested and overlapping PCR reactions. Primers are designated A-G.Primers A and B were used to amplify a 150 base pair fragment from theβ-chain of the IA^(q) and cDNA and incorporating a collagen II (CII)peptide. Primer A is homologous to the leader sequence of the β-chainand encodes a Sal I restriction site to facilitate subcloning of thefinal PCR product into the pCMV8 expression vector. Primer B hashomology to the sequence encoding the first three amino acids of the β1domain of the β-chain and a region of non-homology to encode the CIIpeptide (amino acids 257-269, CII 257-269). The PCR product from thisreaction was purified as described for the α-chain and re-amplified withprimer A and primer C. Primer C has homology to the 3′ region of the A-BPCR product plus a sequence of non-homology encoding the rest of CII257-269 and part of a linker and thrombin cleavage site. This reactiongenerated the A-C PCR product. In a separate reaction, primers D and Ewere used to amplify the extracellular portion (β1 and β2 domains) fromthe IA^(q) cDNA. Primer D has homology to the β1 domain and to the 3′end of the A-C PCR product. Primer E has homology to the end of the β2domain and a region of non-homology corresponding to the hinge region ofIgG₃. This PCR product (D-E) was gel purified as described. To generatethe F-G PCR product, cDNA was prepared from a murine plasma cell knownto produce an immunoglobulin of the IgG₃ subclass (BP107.2.2, ATCC).Total RNA was made using RNazol (Teltest Inc., Friendsworth, Tex.)according to the manufacturer's directions. Oligo dT was used to primethe cDNA reaction using the Superscript Preamplification System (GibcoBRL, Gaithersburg, Md.) according to the manufacturer's directions. Onetwentieth of the cDNA reaction was amplified with primers F and G togenerate a PCR fragment (F-G) encoding the hinge, CH2 and CH3 domains ofthe IgG₃ molecule. Primer F has homology to the hinge region of IgG₃ andhomology to the 3′ region of the D-E PCR product. Primer H has homologyto the CH3 domain and encodes a Sma I restriction site for subcloningthe final PCR product into the expression vector. The F-G PCR productwas purified and amplified together with the D-E PCR product usingprimers D and G to generate the PCR product D-G. This product waspurified on a gel, and annealed with the A-C PCR product using primers Aand G. The final 1500 base pair fragment was purified on a gel, digestedwith Sal I and Sma I and ligated into the pCMV8 expression vector asdescribed for the α-chain. The sequences of the primers are:

A. 5′ CBGTCGACGGATGGCTCTGCAGAT 3′ (SEQ ID NO: 8)

B. 5′ GGGGCCTTGTTCGCCTTTGAAGCCAGCAATACCCAGCTCGGAGTTTCCGCCCTC 3′ (SEQ IDNO: 9)

C. 5′ GCCCCGTGGCAGTAGTGAGCCACCACCTCCGGGGCCTTGTTCGCC 3′ (SEQ ID NO: 10)

D. 5′ GAACAAGGCCCCGGAGGTGGTGGCTCACTAGTGCCACGGGGCTCT 3′ (SEQ ID NO: 11)

E. GTATTCTAGGCTTGCTCCGGGCAGA 3′ (SEQ ID NO: 12)

F. TCACTGTGGAGTGGAGGGCACAGTCCGAGTCTGCCCGGAGCAAGC 3′ (SEQ ID NO: 13)

G. TrCCCGGGTCATTTACCAGGGGAGCG 3′ (SEQ ID NO: 14)

As shown by the preceding description and examples, the fusion proteinof the present invention can be used in a variety of differentapplications, both in suppression of T cell functions and enhancement ofT cell functions. The specificity of the present invention isparticularly useful since the fusion protein is loaded or complexed to apeptide which, together with the MHC, recognizes T cell clones bearingspecific TCR.

14 30 base pairs nucleic acid single linear unknown 1 GCGCATCGATATGGTACCGT GCACGCTGCT 30 29 base pairs nucleic acid single linearunknown 2 CCCTGGGCAC CCATCTCAGG GTGAGGGGC 29 28 base pairs nucleic acidsingle linear unknown 3 CCTGAGATGG GTGCCCAGGG ATTGTGGT 28 30 base pairsnucleic acid single linear unknown 4 AAGCATTCTA GATCATTTAC CAGGAGAGTG 3026 base pairs nucleic acid single linear unknown 5 AAAGATCTAG GATGCCGCGCAGCAGA 26 26 base pairs nucleic acid single linear unknown 6 AACTGCAGAGGATGCCGCGC AGCAGA 26 26 base pairs nucleic acid single linear unknown 7AACCCGGGTT AAGTCTCTGT CAGCTC 26 24 base pairs nucleic acid single linearunknown 8 CBGTCGACGG ATGGCTCTGC AGAT 24 54 base pairs nucleic acidsingle linear unknown 9 GGGGCCTTGT TCGCCTTTGA AGCCAGCAAT ACCCAGCTCGGAGTTTCCGC CCTC 54 45 base pairs nucleic acid single linear unknown 10GCCCCGTGGC AGTAGTGAGC CACCACCTCC GGGGCCTTGT TCGCC 45 45 base pairsnucleic acid single linear unknown 11 GAACAAGGCC CCGGAGGTGG TGGCTCACTAGTGCCACGGG GCTCT 45 25 base pairs nucleic acid single linear unknown 12GTATTCTAGG CTTGCTCCGG GCAGA 25 45 base pairs nucleic acid single linearunknown 13 TCACTGTGGA GTGGAGGGCA CAGTCCGAGT CTGCCCGGAG CAAGC 45 26 basepairs nucleic acid single linear unknown 14 TTCCCGGGTC ATTTACCAGG GGAGCG26

This has been a description of the present invention, along with apreferred method of practicing the present invention. However, theinvention itself should only be defined by the appended claims whereinwe claim:
 1. A fusion protein comprising a plurality of MHC class Imolecules complexed to an immunoglobulin constant domain, and a selectedantigen associated with said plurality of MHC molecules, wherein saidimmunoglobulin constant domain includes a portion that can bind to an F,receptor, said fusion protein stimulating T cells in vivo against saidselected antigen.
 2. The fusion protein of claim 1 wherein said selectedantigen is selected from the group consisting of a viral antigen, atumor antigen, an autoantigen, a bacterial antigen and a fungal antigen.3. The fusion protein of claim 1, wherein said fusion protein comprisestwo MHC molecules.
 4. The fusion protein of claim 1, wherein saidimmunoglobulin constant domain consists essentially of the CH3 domain.5. The fusion protein of claim 6, wherein said immunoglobulin constantdomain further comprises the CH2 domain and the hinge region.
 6. Afusion protein comprising a plurality of MHC class I molecules complexedto an immunoglobulin constant domain, a β2-microglobulin moleculecovalently attached to said plurality of MHC molecules via a peptidetether, and a selected antigen associated with said plurality of MHCmolecules, wherein said immunoglobulin constant domain includes aportion that can bind to an F_(c) receptor, said fusion proteinstimulating T cells in vivo against said selected antigen.
 7. The fusionprotein of claim 6, wherein said fusion protein comprises two MHCmolecules.
 8. The fusion protein of claim 6, wherein said immunoglobulinconstant domain consists essentially of the CH3 domain.
 9. The method ofclaim 8, wherein said immunoglobulin constant domain further comprisesthe CH2 domain and the hinge region.